1. Technical Field
The present invention relates to methods and compositions for treating diseases involving disruption of the glycocalyx, inflammation, and oxidative damage. More specifically, the present invention relates to methods and compositions for treating cardiovascular disease (CVD).
2. Background Art
The existence of the glycocalyx, a thin layer at the endothelial surface was discovered about 40 years ago (1966 Fed Proc 25:1773-1783). However, the significance of this structure was not recognized, partly because it is destroyed upon conventional tissue fixation and not seen in most light microscopic examinations. The glycocalyx is a protective lining at the surface of the endothelium found in every healthy blood vessel; it is made of proteoglycan (PG), a complex network of protein (glycoprotein) and disaccharide sugar (glycosaminoglycan (GAG)). This complex network (originating from plasma and vessel wall) forms a dynamic layer between the flowing blood and the endothelium, continuously changing in thickness depending on shear or blood flow pressure. Thus, the shear generated by blood flow regulates the balance between biosynthesis and shedding of the various glycocalyx components. The core protein groups of this layer are syndecans and glypicans promiscuously bound with different GAG, including heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan (or hyaluronic acid). In the vasculature, heparan sulfate represents roughly 50-90% of the total amount of PGs followed by chondroitin sulfate with a typical ratio of 4:1, respectively (2007 Pflugers Arch; 454: 345-359).
The glycocalyx can also be found on the apical portion of the microvilli within the digestive tract, especially within the small intestine. It creates a meshwork 0.3 micrometers thick and consists of acidic mucopolysaccharides and glycoproteins that project from the apical plasma membrane of epithelial absorptive cells It provides additional surface for adsorption and includes enzymes secreted by the absorptive cells that are essential for the final steps of digestion of proteins and sugars.
Each cell is surrounded by a glycocalyx. The glycocalyx layer of conjoined cells of a tissue form a glycocalyx layer of a tissue's surface and form a barrier. Once disrupted, the underlying cell is susceptible to disruption and immune attack by macrophages and the like. The glycocalyx of endothelial cells, such as the endometrium, the inner surface of the lungs, the microvilli of the kidney, the pancreas, etc., form a cellular seal that cannot be disrupted.
Further, the glycocalyx at the cellular level supports the structural and functional integrity of the glycoproteins and other biomolecules passing there through. Biomolecules that form channels, receptors, and other functional components of the cell membrane structurally and functionally coexist with and through the glycocalyx. Disruption of the glycocalyx results in disruption of the structure and function of those biomolecules, thereby disrupting the structure and function of the cells, as well as the tissues, and organs comprised of those cells.
Other generalized functions effected by status of glycocalyx include protection (it cushions the plasma membrane and protects it from chemical injury), immunity to infection (it enables the immune system to recognize and selectively attack foreign organisms), defense against cancer (changes in the glycocalyx of cancerous cells enable the immune system to recognize and destroy them), transplant compatibility (it forms the basis for compatibility of blood transfusions, tissue grafts, and organ transplants), cell adhesion (it binds cells together so that tissues do not fall apart), inflammation regulation (glycocalyx coating on endothelial walls in blood vessels prevents leukocytes from rolling/binding in healthy states), fertilization (it enables sperm to recognize and bind to eggs), and embryonic development (it guides embryonic cells to their destinations).
Today, the glycocalyx is recognized as a key structure for maintaining vascular wall integrity and proper function of many organs. Disruptions in the glycocalyx can be due to contact with fluid flow. A thick glycocalyx indicates the absence of plaque, found at straight flow and high shear areas. A thin glycocalyx promotes plaque buildup, especially where there is whirlpool blood flow with low shear in vascular bends. Plaques are essentially patches that cover tiny gaps to maintain osmotic balance of membranes. The tiny gaps in the membrane leak electrolytes both into (Na+Cl—, Ca+, HCO3) and out (K+, PO4-, Mg+) of cells which can lead to a number of conditions. Disruptions can also be caused by the presence of oxidants or debris in adjacent fluid.
Any disruption or decrease in thickness of the glycocalyx can result in many different conditions, including chronic vascular disease (2010 Cardiovascular Research. Volume 87, Issue 2 pp. 300-310). For example chronic stagnant blood flow, common in bifurcated sections of the arteries, triggers glycocalyx shedding and plaque formation. In the heart, disrupted glycocalyx in the coronaries result in poor blood flow (coronary perfusion); at the arteriolar level, a damaged glycocalyx slows down blood flow and decreases nitric oxide (NO) production creating constrictive vessel; and, at the capillary level, disrupted glycocalyx reduces blood flow to tissues or muscles. In addition, the glycocalyx harbors a wide array of enzymes that regulate proper blood flow including superoxide dismutase (SOD), an enzyme which neutralizes reactive oxygen species (ROS); antithrombin (AT-III), a natural anticoagulant (blood thinner); and, lipoprotein lipase (LPL), an enzyme that releases triglycerides from chylomicrons and very low-density lipoproteins (VLDL) for energy. See FIG. 1.
In case of cardiac ischemia/reperfusion injury (heart muscle damage due to blood flow obstruction, then re-establishment of blood supply), disrupted glycocalyx results in coronary constriction, poor blood flow, and edema. However, pre-treatment of the heart with antithrombin reduces glycocalyx shedding and restores coronary functions (2009 Cardiovascular Research, Volume 83, Issue 2, pp. 388-396).
Other more general consequences of a disrupted glycocalyx include osmotic gradient shifts, leakage between cells (such as vascular, kidney, and lung cells), macrophage infiltration and inflammation, and tissue dysfunction. Eventually, glycocalyx dysfunction can lead to blockage of flow in vasculature, the kidneys, the pancreas, and other organs and tissue.
CVD is the leading disease killer in the world and because of its complexity and manifested clinical sequalae, it continues to be the main subject in pathology research. Although members of the CVD family are totally different in clinical presentations, they are basically atherosclerosis related and share a common feature, which is vascular damage, particularly to the endothelial glycocalyx. Once the vasculature is damaged, the thromboembolism cascade ensues. Thromboembolism as a process leading to the formation of thrombus (blood clot); once this thrombus dislodges from its origin, it forms an embolus, which flows downstream in the blood vessel tree as a thromboembolus and clogs up blood flow. A thrombus is a solid mass consisting of platelets, fibrin and blood components. An embolus is a piece of thrombus broken free and carried into the bloodstream. Thromboembolus is a floating embolus that becomes lodged and blocks blood flow, which is the fatal component in CVD.
The blood pressure generated by the pumping heart fluctuates and blood flow particularly slows down at arterial forks and bends, notably in the coronary arteries. High fat diet increases blood viscosity and further stagnates blood flow; this stagnation creates low shear and consequently shedding or disruption of the endothelial glycocalyx. Glycocalyx thickness range from 2 to 3 μm in small arteries to 4.5 μm in carotid arteries (2007 J Vasc Res 44:87-98) and shedding or damage to this layer decreases protective shield leading to leakage of nutrients (extravasation) and tissue edema, loss of nutritional blood flow, and an increase in coagulability due to platelet and leucocyte clumping (adhesion). Thus, protection and/or restoration of the endothelial glycocalyx presents a promising therapeutic target both in an acute critical care setting and in the treatment of chronic vascular disease. Drugs that can specifically increase the synthesis of glycocalyx components, refurbish it, or selectively prevent its enzymatic degradation are not currently available. (2010 Cardiovascular Research, Volume 87, Issue 2, pp. 300-310).
Under inflammatory conditions the integrity of the endothelial glycocalyx deteriorates to varying degrees particularly during generalized inflammatory responses, but glycocalyx could regain its original thickness after proper treatment of inflammatory condition (2008 Circulation Research, Volume 102, Issue 7, pp. 770-776). Thus, therapeutic strategies can be directly aimed at preserving, supporting, or reconstituting the glycocalyx structure or strategies either indirectly by down regulating inflammatory processes or directly by inhibition of glycocalyx degradation with antioxidants (2006 American Journal of Physiology: Heart and Circulatory Physiology, Volume 290, Issue 6, pp. H2247-H2256). An example of an anti-inflammatory drug is etanercept (Enbrel®), which inhibits TNF-α, and reduces the shedding of glycocalyx constituents, coagulation activation, and functional vessel function in humans (2009 Atherosclerosis, Volume 202, Issue 1, pp. 296-303).
Another approach is antithrombin therapy, since thrombin is known to cleave the syndecan component of glycocalyx (2009 Circulation Research, Volume 104, Issue 11, pp. 1313-1317). Indeed, antithrombin therapy protects glycocalyx from TNF-α and ischemia/reperfusion-induced shedding in hearts (2009 Basic Research in Cardiology, Volume 104, Issue 1, pp. 78-89; 2010 Shock, Volume 34, Issue 2, pp. 133-139), which is accompanied by reduced post-ischemic leukocyte adhesion in hearts, reduced vascular permeability, reduced coronary leak, and reduced interstitial edema (2009 Basic Research in Cardiology, Volume 104, Issue 1, pp. 78-89).
CVD includes a family of diseases affecting both arteries and veins: diseases in the arteries include coronary heart disease (CHD), myocardial infarction (MI), stroke, hypertension, atrial fibrillation, congestive heart failure (CHF), congenital heart condition, and peripheral arterial disease (PAD); diseases in the veins include venous thrombosis, deep venous thrombosis (DVT), and pulmonary embolism (PE).
CHD results from the effects of atherosclerotic plaque formation in coronary arteries. The reduction in blood supply to the heart muscles reduce the heart's efficiency and can cause heart failure. One of the first and major symptoms of this condition is angina (chest pain caused by reduced blood flow to the heart muscle).
Commonly known as heart attack, MI is the irreversible necrosis of heart muscle due to prolonged interruption of blood supply (ischemia). The heart requires constant supply of oxygen and nutrients; if one of the arteries or branches becomes blocked suddenly, the heart is starved of oxygen, a condition called “cardiac ischemia.” If cardiac ischemia lasts too long, the starved heart tissue dies, which is called heart attack (MI), literally, “death of heart muscle.”
Stroke occurs when brain cells die owing to a lack of blood supply, which may be classified as ischemic or hemorrhagic: ischemic stroke involves decreased blood supply to parts of the brain, leading to brain cell death and thus brain dysfunction; hemorrhagic stroke is due to rupture of blood vessels or abnormal vascular structure, causing accumulation of blood in a part of the brain. The majority of strokes (80%) are ischemic in nature.
Hypertension or high blood pressure is defined as a condition wherein the pressure of the blood flowing through blood vessels remains high for a prolonged period irrespective of the body's need. An increased blood pressure leads the heart to work harder, which makes the heart and arteries more susceptible to injury. Hypertension further increases the risk of incidents such as heart attack, heart failure, and atherosclerosis.
Cardiac arrhythmias are heart rhythm problems, which occur when heartbeats are not well coordinated owing to improper electric impulses. This may cause the heart to beat too fast (tachycardia) or too slowly (bradycardia). Arrhythmias are generally harmless and momentary, but frequent rhythm disturbances increase the risk of stroke and CHF. Atrial fibrillation is the most common sustained arrhythmia
CHF is a condition wherein the heart fails to supply blood to the various parts of the body. This can be due to narrowed arteries, MI, heart valve disease, high blood pressure, cardiomyopathy, or congenital abnormalities.
PAD is a vascular disorder in which the thickening of arteries causes reduction in blood flow to limbs, leading to intermittent leg pain while walking. The disease is an indicator of atherosclerosis. It leads to sores (that do not heal) and gangrenes.
DVT is a blood clot that usually forms in the deep veins of the lower leg or arm, which can block the venous return. A DVT may cause leg pain or swelling, but can also present no symptoms. DVT is not usually life threatening, but it can be if the blood clot breaks loose and lodges into the lungs. This is known as a PE.
Historically, cardiovascular therapeutic drugs do not, nor are they intended to focus on the cause of CVD, but are focused on developing medicines that target the symptoms of CVD. Strategies currently existing in the marketplace are the development and marketing of symptom-targeted drugs while incidences of CVD still continue to rise.
There is an array of symptom-targeted drugs currently marketed against CVD, including cholesterol-lowering drugs such as statins and fibrates for CHD; diuretics, ACE inhibitors, ARBs, calcium inhibitors, and β-blockers for hypertension; and, anticlotting drugs such as anticoagulants (e.g., heparin, rivaroxaban, low molecular weight heparin, dabigatran etexilate mesylate, bivalirudin, coumadin, abciximab, eprifibatide, tirofiban), antiplatelets (e.g., clopidogrel bisulfate, prasugrel, ticagrelor, cilostazol, aspirin, terutroban, dipyridamole), and fibrinolytics (e.g., tissue plasminogen activator (tPA), streptokinase) for stroke. However, these therapies at best mask and treat the symptoms (hypertension, lipidemia, clotting) of CVD, and not the root causes. The therapies decrease levels of fibrin or platelets but do not bust or dissolve clots, they cause either excessive bleeding at high doses or clotting at low doses, and they can decrease Vitamin K levels leading to poor calcium control and heart calcification and osteoporosis. Furthermore, some drugs such as statins have risk of serious side effects such as liver damage, type 2 diabetes, prostate cancer, memory loss, confusion, and dementia.
There are also many other therapeutics existing that merely treat symptoms as opposed to root causes of diseases other than CVD. For example, nitroglycerin is administered for angina symptoms such as chest pain in order to open blood vessels and improve blood flow. It is not administered to treat the underlying cause of why the blood vessels are constricted in the first place. Anti-inflammatories (such as aspirin, ibuprofen, and naproxen (NSAIDS—non-steroidal anti-inflammatory drugs)) are administered in order to reduce inflammation or swelling in the body and relieve pain. They are not administered to treat the underlying cause of why the inflammation is present. Analgesics, especially narcotic analgesics (morphine, codeine, oxycodone, and other opiates), are administered to relieve the symptom of pain or severe pain. They are not administered to treat the underlying cause of the pain. One of the few existing therapeutics that treats an underlying cause is antibiotics, which are administered to kill or inhibit the growth of bacteria in the body. The bacteria themselves can present a whole range of symptoms including pain, irritation, and inflammation that go away once the source is eliminated.
There remains a need for a method of restoring and/or maintaining the integrity of the protective glycocalyx lining of the endothelial vessel wall against atherogenic insults to treat CVD and other diseases. There also remains a need for treating the root causes of CVD and other diseases.